Role of constitutive and induced defences in the resistance of unripe mangoes to Colletotrichum

Transcription

1 Role of constitutive and induced defences in the resistance of unripe mangoes to Colletotrichum gloeosporioides Nimal Adikaram, Ganga Sinniah, Chathurika Karunanayake and Charmalie Abayasekara Department of Botany University of Peradeniya Sri Lanka

2 Anthracnose (Colletotrichum gloeosporioides) and the stem-end rot (Lasiodiplodia theobromae) are two diseases responsible for most of the postharvest losses in ripe mango.

3 Immature mangoes are resistant to both C. gloeosporioides and the stem-end rot (SER) pathogen, Lasiodiplodia theobromae. However, the resistance declines during However, the resistance declines during ripening and anthracnose and stem-end rots occur.

4 Mango fruit defence Immature mango fruits have evolved a formidable defence system comprising constitutive, - antifungal resorcinols in the latex and perhaps the tissue too. - antifungal gallotannins in the peel tissue. - chitinase in the latex (distributed in the peel in a fine net work of minute canals).

5 Substituted antifungal resorcinols OH OH OH OH (CH 2 ) 11 CH (CH 2 ) 14 CH CH 3 (CH 2 ) 3 5- pentadecyl resorcinol CH 3 5- (12- cis-heptadecenyl) resorcinol Cojocaru et al. (1986) Dichloromethane extract HPLC showed two additional peeks one with general features of resorcinols and appears a resorcinol and the other a resorcinol derivative, Sri Lankan cultivars.

6 Resorcinol content in unripe mango fruit peel Resorcinol level (µg/g FR) Cultivar 5-(12- cis-heptadecenyl) 5-pentadecyl AR19 AR21 resorcinol resorcinol Gira (R)* Karuthacolomban (R) Rata (R) Kohu (M) Petti (S) Willard (S) *Resistant (R) to anthracnose Moderately susceptible (M) Susceptible (S)

7 Resorcinol levels in fruit peel at different stages of ripening in a resistant cultivar (`KC ) Resorcinol amount (micro grams/g fresh wt.) Days after harvest KC 5-(12 cic heptadecenyl) resorcinol KC 5-pentadecyl resorcinol KC AR21 CLR Break 20 % OR 50 % % decline, 5-(12-cis-heptadecenyl) resorcinol 85% 5-pentadecyl resorcinol 25% AR 21by approximately 80 %

8 Resorcinol levels in the peel of fruit of susceptible cultivar Resorcinol conc centration Micrograms/ g fresh wt unripe ripe Cultivar 'Willard ' 5-pentadecyl resorcinol 5-(12 cic heptadecenyl) resorcinol AR21

9 Aqueous methanol extract A mixture of several related Gallotannins Prominent inhibition at Rf 0.00 (Karunanayake et al., 2011)

10 MeOH CH 2 Cl 2 MeOH CH 2 Cl 2 MeOH CH 2 Cl 2 Cladosporium sp. Colletotrichum gloeosporioides Lasiodiplodia theobromae Gallotannins inhibit C. gloeosporioides and not L. theobromae.

11 Gallotannin antifungal activity in different mango cultivars Inhibition area on TLC (mm 2 ) Gira KC Rata Kohu Petti Willard 54% decline in activity in susceptible cv. Petti during ripening. 21% decline in the resistant cv. KC KC > Gira > Rata > Kohu > Petti > Willard Cultivars Gallotannin activity in unripe peel extracts Gallotannin activity in ripe peel extracts Lesion area/ mm Inhibition area on TLC/ mm2 Gallotannins content and disease level are negatively correlated (62%) Days 0 Lesion area Inhibition area

12 Mango latex When removed from the fruit, mango latex separates in to an oily phase and an aqueous phase. The oily phase contains antifungal resorcinols & the aqueous phase chitinase. Oily phase Aqueous phase

13 Chitinase activity in the aqueous phase of mango latex 2 min 180 min

14 Lasiodiplodia theobromae

15 Chitinase activity in the aqueous phase of latex in selected mango cultivars a c d e g b h f Activity in un nits/10micro litres Gira KC Rata Kohu Petti Willard (a) Gira (b) Karuthacolomban (c) Kohu, (d) Petti (e) Rata (f) Willard (g) Papaya latex (+positive control) (h) Phosphate buffer (- control) Rata & Kohu >> Willard & Petti >> Gira & KC

16 Cultivar Resistance/Susceptibility Anthracnose SER Relative gallotannin content Latex chitinase activity (units/ l) Ambalavi MR S 0.083±0.025 Dilpasan S R ±0.010 Gira R S 355/207*(42%) 0.094± KC R S 295/231 (21.9%) Kohu MR R 271/171 (36.9%) 0.123± Malvana R R ±0.032 Neelam R R ±0.031 Petti S R 321/146 (54.3%) 0.189± Rata MR R 318/178 (44%) Seylum MR R ±0.019 Willard S R 148/100 (33%) -- Volume of latex 0.029± ± ± ±

17 Volumes of latex cultivar Volume, ml KC (R) Rata (MR) Oil phase % Oil phase Interiediate Aqueous % Aqueous phase phase Total volume 1.5± ± ± ± ± ± ± ± ± ± ± ±0.46 Willard 0.43± ± ± ± ± ±0.39 (S) Kohu (MR) Petti (S) Gira (R) % The % of the oil phase is correlated with the amount of resorcinols (Hassan, 2006). The % of the oil phase was greater in more resistant cultivars

18 Anthracnose development in fruits in which latex was retained Lesion area (mm 2 ) Willard Anthracnose was significantly (P<0.05) less in latex-retained fruits of the susceptible cv Days after inoculation Fruits from w hich latex w as drained Fruits from w hich latex w as not drained Lesion area (mm2) Karuthacolomban Days after inoculation Anthracnose was less in fruits from which latex was not drained, but not significant (resistant cv.) Fruits from w hich latex w as drained Fruits from w hich latex w as not drained

19 Cultivar `Willard Latex drained Control Chitinase activity, units/g FW Day 1 Day 3 Day `KC Latex drained Control

20 Induced defence responses

21 Mango peel tissue responds to C. gloeosporioides by producing Reactive Oxygen Species (O 2- ) & H 2 O 2, increased peroxidase and chitinase activity, increased phenols during early hours. No phytoalexins induced.

22 Germination and appressoria formation by C. gloeosporioides on unripe mango fruit a b L GT NA PP MA S a BC 9 h b 18 h IP A BC A Germination 3h after inoculation Appressoria formation 6 h Melanization of appressoria 9-12 h Infection hyphae 24 h 48 h

23 O 2- generation in epidermal cells of mango peel following inoculation with C. gloeosporioides Karuthacolamban a 12 h 24 h b Willard c d Epidermal peels stained with 0.1% Nitroblue tetrazolium (NBT)

24 H 2 O 2 generation in epidermals cells following inoculation with C. gloeosporioides a b First observed 9-12 h after inoculation; peels were stained with diaminobenzidine (DAB)

25 Autofluorescence and cell browning 12 ah 24 h 48 h Autofluorescence Cell browning Accumulation of phenols Cell death HR??

26 Chitinase activity in the peel of mango cultivar Karuthacolamban and Willard following inoculation with C. gloeosporioides. 2 Chitinas ase activity (U) Time after inoculation (h) Karuthacolamban Willard Infected Healthy Induced isozymes 59.4 & 44.5 kda

27 100 Intreaction site (%) Time after inoculation (h) Resistant (KC) Susceptible (Willard) Interaction site (%): Percentage of appressoria showing blue staining in the target cells/surrounding in a field of vision.

28 Differentially expressed genes in the interaction between mango fruits and C. gloeosporioides Expressed bands Differentially expressed bands Karutha Willard colamban 6 1 Up regulated 2 2 Down regulated 3 7

29 Conclusions Resistance of mango is due to a combination of the constitutive defences - antifungal resorcinols, gallotannins and the enzyme chitinase. In addition, some defence responses appear to be induced when the fruit is challanged with C.gloeosporioides. Resistant cultivars have higher levels of one or more of the constitutive defences, and also induce higher levels new defences. Latex plays a defensive role. Fruit resistance to anthracnose may be enhanced by latex management.

30 Treatments that retain/enhance constitutive defences and induce new defences will help disease management. Some of these information (e.g. gallotannin Some of these information (e.g. gallotannin content, chitinase activity) would be useful in selection of resistant cultivars.

31 Acknowledgement Australian Centre for International Agricultural Research (ACIAR)

32

33 H-T 11 A, OPA11 H-T 11 A, OPB6 H-T 11 C, OPD KU1 KDD4 KD1 KDD5 KD2 Differential display profile of RNA samples isolated at different times following inoculation with C. gloeosporioides from unripe mango cultivars Karuthacolamban (K) using the anchor primer HT11M (Where H- AAGC, M can be either G, A, or C). in combination with random primers given at the top. DD- differentially displayed band, U-up regulated bands, D- down regulated bands.